The present invention relates to a fluorescent pattern reading apparatus and, more particularly, to a fluorescent pattern reading apparatus capable of reading a fluorescent pattern by appropriately varying sensitivity to fluorescent patterns of various samples in accordance with characteristics of fluorescence intensity, background light, etc.
Generally, electrophoresis analysis of samples labeled with a radioactive isotope is employed for analysis of the structures of various genes, including DNA sequencing (determination of the sequence of bases of the gene), mass analysis of proteins such as amino acids, and analysis of the structure of a polymer. The electrophoresis analysis involves performing electrophoresis using a gel containing fragments of a sample labeled with a radioactive isotope, transcribing a distribution pattern of fragments developed by the electrophoresis to an X-ray film, and then performing analysis of the distribution pattern.
On the other hand, in recent years, as technology of light sources such as the laser has advanced, there has been developed the electrophoresis analysis of samples labeled with a fluorescent substance, in place of the radioactive isotopes, by using the fluorescence method. The electrophoresis pattern reading apparatus using the fluorescence method presents the advantage that no dangerous and expensive radioactive isotopes are required. In order to allow the electrophoresis pattern reading apparatus using the fluorescence method to achieve a signal-to-noise ratio (a S/N ratio) equal to the electrophoresis analysis using the radioactive isotopes, high technology of an optic system and signal processing technology are required.
Description will be made of a representative example of the electrophoresis pattern reading apparatus by taking a DNA sequencing apparatus as an example. When the DNA sequencing is performed by using the DNA sequencing apparatus, a sample containing a DNA whose structure is to be determined is fragmented by controlling a reaction rate of a chemical reaction specific to the site of each base with a restriction enzyme and labelling the resulting fragments with a fluorescent substance. The fragments are different in length and each of the fragments contains a certain particular base selected from four different bases, i.e. adenine (A), cytosine (C), guanine (G) and thymine (T), at its cut terminal. The fragments of the DNA so fragmented as to contain A, C, G and T are separated in accordance with the lengths of the fragments by electrophoresis, so that the structure of the DNA is determined by performing the electrophoresis to separate the fragments, radiating the separated fragments with laser light, exciting the fluorescent substance labelled on each fragment, measuring the distribution of intensity of the fluorescence emitted from the fluorescence substance, and reading the sequence of the bases.
FIG. 16 is a schematic diagram showing an example of the distribution of the DNA fragments provided by performing the electrophoresis. As the distance of migration by the electrophoresis varies with lengths of the DNA fragments (the difference of their molecular weights), the fragments having the same molecular weights gather together as time elapses, thereby forming an electrophoresis pattern 70 as shown in FIG. 16. The electrophoresis pattern 70 is formed by developing the fragments into each band 66 in accordance with their molecular weights, and the pattern is formed as a whole by developing the sample into each of the bands 66 on lanes 71, 72, 73 and 74 corresponding to each of the four bases. The amount of the sample required by each band is extremely small, as low as 10-16 mol. As difference in molecular weight occurs among the DNA fragments for the bases A, C, G and T by the molecular weight corresponding to one base or more, the distances migrated by the electrophoresis varies with the bands on lanes 71, 72, 73 and 74 for the respective bases. Hence, the bands 66 in the lanes 71, 72, 73 and 74 for the respective bases A, C, G and T are not theoretically disposed transversely in a row with each other. For the DNA sequencing, the sequence of the DNA is analyzed by scanning the order of the bands 66 from the bottom in each of the lanes 71 to 74 for the respective bases A, C, G and T.
As described hereinabove, the analysis by means of the electrophoresis method can be applied to the DNA sequencing apparatus for analyzing the sequence of the bases of a DNA. It can further be noted that the analysis by means of the electrophoresis method can likewise be applied to electrophoresis of other samples. In this case, the sample to be analyzed is subjected to electrophoresis, thereby reading the distribution pattern developed by the electrophoresis. By performing the electrophoresis of the sample to be analyzed, the sample is separated into bands in accordance with its molecular weights or with a charge amount of the sample in a solvent, and the distribution of the resulting bands is read, thereby determining the difference in molecular weight of the samples from the extent to which the bands are distributed. And the molecular weight can be estimated and the presence or absence of a given molecule can be determined by measuring the distance migrated by the electrophoresis of the fragments of the sample and determining the presence or absence of the band in a predetermined position.
In the analysis by electrophoresis, a sample labeled with a fluorescent substance is first poured into a gel serving as a base and the gel is then subjected to electrophoresis. After the completion of the electrophoresis, the gel is provided with a distribution pattern in which the bands are distributed in accordance with the difference in molecular weight among the fragments of the sample, and the distribution of the bands are then measured. The measurement of the distribution of the bands is performed by radiating the electrophorezed gel with a light, such as laser light or lamp light serving as light for exciting a fluorescent substance and measuring the distribution pattern by sensing the fluorescence excited from the gel by a photoelectrically converting element. As the gel, there may be employed, for example, polyacrylamide gel or agarose gel. The sample may be labeled with the fluorescent substance prior to electrophoresis or by drying the gel subsequent to electrophoresis or after the transcription of the sample to a thin film filter from the gel, etc.
As an example of the electrophoresis apparatus of this type using the electrophoresis detecting method, there may be mentioned an electrophoresis apparatus as disclosed in Japanese Patent Laid-open Publication (kokai) No. 62,843/1986 (corresponding to U.S. Pat. No. 4,675,095).
Description will now be made specifically of the electrophoresis apparatus using the fluorescence detecting method.
FIG. 12 is a perspective view showing an outlook of conventional electrophoresis device. As shown in FIG. 12, the conventional electrophoresis device comprises an electrophoresis and instrumentation unit 51 for carrying out electrophoresis and instrumenting the distribution of fluorescence, a data processor unit 52 for processing data instrumented, and a cable 53 connecting them to each other. The electrophoresis and instrumentation unit 51 has a door 51a and the door 51a is opened to pour a gel functioning as a base for electrophoresis of DNA fragments and then a given amount of a sample to be analyzed. Then the door 51a is closed and a switch is turned on to start up electrophoresis. As electrophoresis has been started up, an operational state is displayed and monitored on a display panel 51b of the electrophoresis and instrumentation unit 51. The data instrumented is then transferred to the data processor unit 52 and is subjected to desired data processing in accordance with the preset programs. The data processor unit 52 comprises predominantly a main body of a computer 54 consisting of a microprocessor, memory and so on, a keyboard 55 from which instructions are given by the operator, a display 56 for display of processing results and states, and a printer 57 for recording the processed results.
FIG. 13 is a block diagram showing the construction of the inside of the electrophoresis and instrumentation unit. As shown in FIG. 13, the electrophoresis and instrumentation unit 51 (FIG. 12) comprises an electrophoresis subunit 63 and a signal processor subunit 64. The electrophoresis subunit 63 further comprises an electrophoresis section 5 in which electrophoresis is performed, a first electrode 2a and a second electrode 2b for applying voltage to the electrophoresis section 5, a support plate 3 for supporting the electrophoresis section 5 and the first and second electrode 2a and 2b, a power supply 4 for applying voltage to the electrophoresis section 5, a light source 11 for generating light to excite a fluorescent substance, an optical fiber 12 for leading the light from the light source 11, a condenser 14 of an optic system for condensing and collecting fluorescence 13 generated by the fluorescent substance, an optical filter 15 for selectively passing the light having a particular wavelength therethrough, and an optical sensor 16 for converting the condensed light into electrical signals. The signal processor subunit 64 further comprises an amplifier 17 for amplifying the electrical signals from the optical sensor 16, an analog-digital converting circuit 18 for converting analog signals of the electrical signals into digital data, a signal processing section 19 for implementing pre-processing of the digital data converted, for example, by addition average processing or the like, an interface 20 for implementing the interface processing for feeding the pre-processed data to an external data processor, and a control circuit 10 for performing the entire control of the electrophoresis and the signal processing. The digital signal OUT is generated from the signal processor subunit 64 and then supplied to the data processor unit 52 (FIG. 12), thereby implementing the data processing such as analysis processing and so on.
Description will now be made of the operation of the electrophoresis device which is constructed in the manner as described hereinabove.
Reference is made to FIGS. 12 and 13. After opening the door 51a of the electrophoresis and instrumentation unit 51, a gel is poured into the electrophoresis section 5 disposed within the unit 51 and thereafter a sample of DNA fragments labeled with a fluorescent substance is poured thereinto. A switch of the display panel 51b is turned on to give an instruction for starting electrophoresis, and then voltage is applied from the first and second electrodes 2a and 2b of the power supply 4 to the electrophoresis section 5, thereby starting up electrophoresis. The electrophoresis allows the sample labeled with the fluorescent substance to be migrated in the lanes 71, 72, 73 and 74, thereby gathering the molecules having the same molecular weights together and forming the bands 66, for instance, as shown in FIG. 16. The molecules having smaller molecular weights are allowed to migrate at a rate faster than those having greater molecular weights so that the former are migrated a distance longer than the latter within the same time unit. The bands 66 are detected in a manner as shown in FIG. 14a by leading light from the light source through the optical fiber 12 and irradiating the gel in the transverse direction of the electrophoresis section 5 on its optical path, thereby forcing the labeled fluorescent substance gathered on the bands 66 of the gel to emit fluorescence 13. The fluorescence generated is so very faint because only a very minute amount of the fluorescent substance is present, as small as approximately 10.sup.-16 mole per band although the amount thereof may vary to some extent with the absorptivity of the fluorescent substance, the quantization efficiency, the intensity of the exciting light and so on. When fluorescein isothiocyanate is used as the fluorescent substance, the fluorescein isocyanate has a peak of the exciting wavelength of the exciting light at 490 nm, a peak of fluorescence at 520 nm, a molar absorptivity at 7.times.10.sup.4 mol.sup.-1 .multidot.cm.sup.-1, and the quantization efficiency of approximately 0.65.
If the fluorescent substance exists in the amount of 10.sup.-16 mole per band, photons of the fluorescence are produced in the order of 10.sup.10 /s when argon ion laser is employed as the exciting light at the output of 1 mW at 480 nm although the light quantity of the fluorescence varies with the thickness of the gel and so on. Further, it is to be noted that the fluorescence spreads in all peripheral directions so that it is difficult to receive the light directly by a general CCD solid state image pick-up element camera or the like.
Referring to the front view as shown in FIG. 14a and to the longitudinally sectional view as shown in FIG. 14b, the electrophoresis section 5 comprises a gel 5a consisting of polyacrylic amide or the like and gel supporting members 5b and 5c made of glass for supporting and interposing the gel 5a from both sides. For example, a sample of DNA fragments is poured into the gel 5a of the electrophoresis section 5 from its upper portion and electrophoresis is carried out by applying voltage to the first electrode 2a and the second electrode 2b (FIG. 12). Light radiated from the light source, for example, laser light, passes through the light path 61 in the gel 5a from the optical fiber 12 and is irradiated to the fluorescent substance on the light path 61. This allows the fluorescent substance present on the light path 61 to be excited to emit fluorescence 13. The fluorescence 13 emitted is led to a substage condenser or light collector 14 of optics consisting of a combination of lenses and then selected by the optical filter 15 after being condensed, thereby converting it into electrical signals by means of the one-dimensionally optical sensor 16. In order to efficiently convert the faint light into electric signals, the image is converted into the electric signals by the optical sensor 16, such as the one-dimensionally optical sensor of the CCD, by optically amplifying the image to 10.sup.4 to 10.sup.5 times by an image intensifier or the like. The electrical signals obtained by the optical sensor 16 are amplified to a desired level by the amplifier 17 and subjected to analog-digital conversion by the analog-digital circuit 18 followed by supply to the signal processing section 19. The signal processing section 19 processes the signals by means of addition-average processing or the like in order to improve a signal-to-noise ratio (a S/N ratio). The data of the digital signals which has been subjected to signal processing is fed to the data processor subunit 52 through the interface 20.
FIGS. 15a and 15b are views describing an embodiment of signals indicative of a fluorescence intensity pattern of the DNA fragments to be transferred from the electrophoresis and instrumentation subunit 51. For instance, as shown in FIG. 15a, as the laser light is irradiated upon the electrophoresis section 5 in which the electrophoresis is performed, the fluorescent substance of the gel present on the light path 61 is excited to emit fluorescence 13. This fluorescence 13 is detected in predetermined detection positions in each lane in the direction of electrophoresis in the course of a lapse of time. This allows the fluorescence 13 to be detected when the bands 66 in each lane pass through the positions on the light path 61, thereby detecting a pattern signal of fluorescence intensity in each lane, as shown in FIG. 15b. Therefore, the pattern signal of the magnitude of fluorescence intensity as shown in FIG. 15b is represented as a pattern signal of fluorescence intensities of the bands 66 in the electrophoresis direction 62.
The data processor unit 52 performs data processing for comparing molecular weights and determining the sequence of DNA from data of the pattern of fluorescence intensity. The sequence of the bases or the like determined by data processing is symbolized and then generated, thereby being displayed on a display screen 56 or printed by a printer 57. The data of the result obtained by data processing may be recorded in magnetic recording media as needed.
It is to be noted herein that, as described hereinabove, the electrophoresis pattern reading apparatus using the fluorescence detecting method is so constructed that the light is irradiated upon the electrophoresis section in the transverse direction from the light source, thereby providing the exciting light. The incidence of the light from the side has the advantage that the sensitivity with which the gel receives the fluorescence through the glass of the gel supporting plate becomes high because the gel is irradiated with the light directly from the light source so that the glass of the gel supporting plate is not exposed to the exciting light from the light source so that the light does not scatter from the exciting light.
However, when for example agarose gel is employed as the gel for the electrophoresis section, a degree of scattering the exciting light in the gel occurs so that it is difficult to keep the size and the strength of the spot light of the light beam of the exciting light uniform over the entire length of the light path 61. In other words, the light intensity is high on the side of the light path 61 which the exciting light strikes while the intensity of the light becomes lower as the light proceeds within the gel. Further, if bubbles were to form at the end portions of the electrophoresis section which are not involved with electrophoresis when the gel is poured prior to the electrophoresis, it is difficult to let the exciting light strike the gel or a large degree of scattering may happen when the bubbles exist on the light path 61 for the exciting light. Furthermore, when the light is irradiated in the transverse direction, all the fluorescent substance-labeled bands located on the light path are caused to emit fluorescence simultaneously. Accordingly, in this case, there must be employed, as an optical sensor for receiving and converging the very faint fluorescence, a combination of an image intensifier and a CCD camera or an expensive one-dimensional optical sensor (a line sensor) of high sensitivity such as a CCD camera so modified as to equivalently increase its sensitivity by cooling the CCD solid state image pick-up element in order to reduce dark current.
It is also to be noted that such an electrophoresis pattern reading apparatus suffers from the disadvantages that it is of a type capable of reading the electrophoresis pattern during the electrophoresis so that it is not suitable for reading fluorescence patterns other than those for determining the base sequence of DNA.